Synthesis of indole thiazole compounds as ligands for the Ah receptor

ABSTRACT

A method of synthesizing aromatic ketone compositions of formula I comprising the step of introducing a double bond into the 5 membered ring of the 4,5-dihydro-1,3-azoles moiety of formula II is disclosed. A method of synthesizing aromatic ketone compositions of formula I comprising the step of ring synthesis of the tetrahydro-1,3-azoles of formula XI is also disclosed.

CROSS-REFERENCE TO RELATED APPLICATION

The application claims priority from provisional patent application Ser.No. 60/356,585, filed Feb. 12, 2002, and U.S. Ser. No. 10/074,102, filedFeb. 12, 2002, all incorporated by reference herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENTBACKGROUND OF THE INVENTION

The Aryl Hydrocarbon Receptor. The aryl hydrocarbon receptor (AhR) is aligand inducible transcription factor mediating a broad spectrum ofbiological processes upon binding to its ligand. Besides induction ofenzymes in the cytochrome P450 family, the receptor appears involvedwith cell proliferation, tumor promotion, immune suppression, vitamin Adepletion, and developmental and reproductive abnormalities (Fletcher,et al., Toxicol. Sci. 62(1):166-175, 2001; Safe, Toxicol. Let. 120:1-7,2001; Gu, et al., Ann. Rev. Pharmacol. Toxicol. 40:519-561, 2000;Poellinger, Food Add. Contam. 17(4):261-266, 2000; Schmidt andBradfield, Ann. Rev. Cell Dev. Biol. 12:55-89, 1996; Whitlock, et al.,Drug Metabol. Rev. 29:1107-1127, 1997). The liganded receptor alsocauses cell cycle arrest, apoptosis, adipose differentiation, andanti-estrogen effects (Bonnesen, et al., Cancer Res. 61(16):6120-6130,2001; Elferink, et al., Mol. Pharmacol. 59(4):664-673, 2001; Shimba, etal., J. Cell Sci. 114(15):2809-2817, 2001; Shimba, et al., Biochem.Biophy. Res. Com. 249(1):131-137, 1998; Safe, supra, 2001; McDougal, etal., Cancer Res. 61(10):3902-3907, 2001; McDougal, et al., Cancer Lett.151:169-179 2000; Elizondo, et al., Mol. Pharmacol. 57(5):1056-1063,2000; Puga, et al., J. Biol. Chem. 275(4):2943-2950, 2000; Alexander, etal., J. Cell Sci. 111(Part 22):3311-3322, 1998). The presence of thereceptor was proposed and evidenced in 1970's (Poland, et al., J. Biol.Chem. 251:4936-4946, 1976). The coding sequence for the receptor wascloned in 1990's and revealed that the AhR is a member of an emergingbasic Helix-Loop-Helix/Pas-Arnt-Sim (bHLH/PAS) transcription factorsuper family (Burbach, et al., Proc. Natl. Acad. Sci. USA 89:8185-8189,1992).

The bHLH/PAS Super Family of Transcription Factors. The bHLH/PAS superfamily includes Drosophila Per, Arnt (Ah receptor nuclear translocator,the dimerization partner of AhR and others), SIM1, SIM2, TRH, ARNT-2,the hypoxia inducible factor-1 (HIF-1α), SRC-1, TIF2, RAC3, MOPs 2-5(Gu, et al., supra, 2000; Hogenesch, et al., J. Biol. Chem.272:8581-8593, 1997; Wilk, et al., Genes Dev. 10:93-102, 1996), andendothelial PAS domain protein (EPAS-1) (Tian, et al., Genes Dev.11:72-82, 1997). These bHLH proteins contain the 300 amino acid PASdomain, composed of two 50 amino acid degenerate direct repeats(Burbach, et al., supra, 1992; Dolwick, et al., Mol. Pharmacol.44:911-917, 1993; Dolwick, et al., Proc. Natl. Acad. Sci. USA90:8566-70, 1993). The basic region is important for DNA binding, andthe HLH and PAS domains are involved in dimerization, and for AhR, inligand binding (Swanson and Bradfield, Pharmacogenetics 3:213-230,1993). The transactivation domains of the AhR and ARNT map to theircarboxyl termini (Jain, et al., J. Biol. Chem. 269:31518-31524, 1994).Members of this super family are master developmental regulators and itis intriguing to speculate similar roles for AhR and ARNT. Besides withAhR, ARNT forms heterodimers also with HIF-1α, PER, SIM, MOP2(Hogenesch, et al., supra, 1997), and EPAS-1 (Tian, et al., supra, 1997)and an ARNT-related protein is postulated to heterodimerize with TRH(Wilk, et al., supra, 1996). This promiscuity of ARNT indicatesAhR-independent roles for ARNT and suggests the possibility of crosstalk between AhR and the other bHLH/PAS signaling pathways.

The Homeostatic Response to Hypoxia: Role of HIF-1a/ARNT-Mediated GeneExpression. Vertebrates require molecular oxygen for vital metabolicprocesses. Homeostatic responses elicited by hypoxia includeerythropoiesis, angiogenesis, and glycolysis. These adaptive responsesserve to increase oxygen delivery or activate alternative metabolicpathways that do not require oxygen in hypoxic tissues. In response tohypoxia, HIF-1α translocate into the nucleus where they formheterodimers with ARNT (Gradin, et al., Mol. Cell. Biol. 16(10):5221-31,1996; Schmidt and Bradfield, supra, 1996). The HIF-1α/ARNT heterodimersbind to hypoxia response elements increasing transcription of genesinvolved in maintaining oxygenation of tissues. The hypoxia-induciblegene products include erythropoietin (EPO), vascular endothelial growthfactor (VEGF), and glycolytic enzymes (Maltepe and Simon, J. Mol. Med.76(6):391-401, 1998).

The Mode of Action of AhR/ARNT Signaling Pathway. The cytoplasmic formof AhR is associated with 2 molecules of heat shock protein (hsp90) andsome other cellular factors (Poellinger, supra, 2000; Whitlock, Ann.Rev. Pharmacol. Toxicol. 30:251-277, 1990). After ligand binding, thehsp90 and the other factors dissociate and AhR is activated. Theactivated AhR then translocates into the nucleus and dimerizes with itspartner ARNT (Probst, et al., Mol. Pharmacol. 44:511-518, 1993).AhR/ARNT heterodimers recognize and bind the so-called xenobioticresponse elements (XREs) found in promoters of AhR controlled genes toalter gene expression (Whitlock, supra, 1990). Another potentialmechanism involves competition between AhR and either HIF-1α and/orEPAS-1 for dimerization with ARNT. Since AhR, HIF-1α and EPAS-1 requiredimerization with ARNT to control the expressions of their target genes,activation of AhR might reduce the availability of free ARNT to such anextent that it becomes rate limiting for other signaling pathways.Decreased availability of ARNT could lead to decreased expression ofvital hypoxia-regulated genes and angiogenesis blockage, for example, byinhibiting HIF-1α signaling (Gradin, et al., supra, 1996; Schmidt andBradfield, supra, 1996).

The Known AhR Ligands. Among the first discovered human-made ligands forthe AhR are the chemicals known as polycyclic aromatic hydrocarbons suchas 3-methylcholanthrene and benzo[α]pyrene. A much more potent andhigher affinity ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), wasdiscovered (Poland and Glover, Mol. Pharmacol. 9:736-747, 1973). Anotherstructural group of compounds, halogenated aromatic hydrocarbons, wasrecognized as the receptor ligands. The compounds with differentstructural characteristics from the groups mentioned were also found tohave binding affinity to AhR. This group is represented by bilirubin(Phelan, et al., Arc. Biochem. Biophy. 357(1):155-163, 1998; Sinal andBend, Mol. Pharmacol. 52(4):590-599, 1997), lipoxin A(4) (Schaldach, etal., Biochem. 38(23):7594-7600, 1999), brevetoxin-6 (Washburn, et al.,Arc. Biochem. Biophy. 343(2):149-156, 1997), diaminotoluene (Cheung, etal., Toxicol. Appl. Pharmacol. 139(1):203-211, 1996), and YH439, athiazolium compound (Lee, et al., Mol. Pharmacol. 49(6):980-988, 1996).Among most of the human-made AhR ligands, TCDD is one of the most potentagents for AhR and is the prototype compound used to study the mechanismof AhR action and dioxin toxicity. The term “dioxins” has been used torefer to any of the PCDDs (polychlorinated dibenzo-p-dioxins), PCDFs(polychlorinated dibenzofurans), or PCBs (polychlorinated biphenyls)that cause the same biological responses, by the same mechanism as TCDD.

The AhR Ligands with an Indole Moiety. The other recognized AhR ligandswith an indole moiety are of special interest. This group consists oftryptamine, indole acetic acid (Heathpagliuso, et al., Biochem.37(33):11508-11515, 1998), indole-3-carbinol and its derivatives(Stephensen, et al., Nutr Cancer Internatl. J. 36(1):112-121, 2000;Chen, et al., Biochem. Pharmacol. 51(8):1069-1076, 1996; Vasiliou, etal., Biochem. Pharmacol. 50(11):1885-1891, 1995; Liu, et al.,Carcinogenesis. 15(10):2347-2352, 1994; Jellinck, et al., Biochem.Pharmacol. 45(5):1129-1136, 1993), and indolo[3,2-b]carbazole (ICZ)(Chen, et al., J. Biol. Chem. 270(38):22548-22555, 1995; Kleman, et al.,J. Biol. Chem., 269(7):5137-5144, 1994). Closely related to ICZ,6-formylindolo[3,2-b]carbazole derived from tryptophan by UV oxidationhas higher affinity than that of TCDD to the receptor (Rannug, et al.,Chem. Biol. 2(12):841-845, 1995; Rannug, et al., J. Biol. Chem.262:15422-15427, 1987). Some of the indole derived AhR ligands displayedtheir interesting properties: binding to the receptor, low toxicity,antiestrogenic and antitumorigenic. Actually, clinical studies have beenlaunched for indole-3-carbinol as an anticarcinogenic andantitumorigenic remedy for patients with high risk of tumorigenesis(Preobrazhenskaya and Korolev, Bioorganicheskaya Khimiya. 26(2):97-111,2000).

Identity of the Endogenous AhR Ligand and Physiological Functions of theAh Receptor System Are not Resolved. Okamoto, et al. (Okamoto, et al.,Biochem. Biophys. Res. Commun. 197:878-885, 1993) observed that exposureof adult male rats to hyperoxia (95% oxygen) caused induction of CYP1A1in the lung and CYP1A1 and 1A2 in the liver. The induction of CYP1A1/1A2is usually associated with the binding of AhR to its ligands. Onehypothesis to explain CYP1A1/1A2 induction by hyperoxia is that anendogenous ligand(s) of the AhR is produced by hyperoxia, whichactivates the transcription of CYP1A1/1A2 genes (Okamoto, et al., supra,1993). Recently two human urinary products were isolated that bind tothe AhR (Adachi, et al., J. Biol. Chem. 276(34):31475-31478, 2001).Whether those products are endogenous ligands or not is undeterminedbecause the identified compounds are indigo, a commonly used fabric dye,and indirubin, an isomer of indigo. Since they were isolated from urine,the question of whether they are urinary excretion products remainsunanswered. Similarly, the bilirubin-related compounds (Phelan, et al.,supra, 1998; Sinal and Bend, supra, 1997) and lipoxin A(4) (Schaldach,et al., supra, 1999) are certainly endogenous in nature but whether theyare the true ligands for the AhR has not yet been resolved. The responseand affinity for the AhR appear to be, in fact, quite low for thesecompounds.

The generation of AhR-deficient mice illustrates possible physiologicalfunctions of the receptor in liver, heart, ovary, and the immune system,even though it is not conclusive at this point (Benedict, et al.,Toxicol. Sci. 56(2):382-388, 2000; Poellinger, supra, 2000; Mimura, etal., Gene. Cell. 2:645-654, 1997; Schmidt, et al., Proc. Nat. Acad. Sci.USA 93:6731-6736, 1996; Fernandez-Salguero, et al., Science 268:722-726,1995). The significance of those findings is that they demonstrate aneed for a functioning AhR signaling pathway in animal physiology. It isprobable that endogenous AhR ligands in animal tissues are involved incarrying out this AhR signaling function.

Importance of Identifying the Endogenous AhR Ligands. Studies withhuman-made AhR ligands on this receptor system greatly advanced ourunderstanding in this system. It is clear, however, that the AhR did notdevelop in an evolutionary sense to react to manufactured chemicalagents. It is reasonable to suspect that there must be an endogenousligand for the AhR, which should be nontoxic at tissue concentrationsnormally encountered in the body, rapidly cleared by metabolism, andutilized to activate the AhR only transiently in a regulatory capacity.Also, evidence shows that the different outcomes of the ligand-receptormediated signaling processes are possible and dependent upon the natureof the ligands. A decisive factor dictating the consequences in theligand-receptor mediated signal transducing systems is the final threedimensional conformation of the liganded receptor assumes because thatconformation determines the ways the liganded receptor interacts withnumerous other factors to transduce signals. Given the amino acidsequence of the receptor, the final three-dimensional structure of theliganded receptor is solely dependent on the structure of the ligand,which ultimately dictates the biological outcomes of the signalingsystem. To completely understand the physiological functions of the Ahreceptor system and the potential therapeutic benefits this system mayoffer, the identification and synthesis of the AhR ligand is an absolutenecessity.

BRIEF SUMMARY OF THE INVENTION

In one embodiment, the present invention is a method of synthesizingaromatic ketone compositions of formula I

wherein:

R₁ may be hydrogen or can be selected from the group consisting of(C₁-C₆)-alkyl and (C₃-C₇)-cycloalkyl, and wherein the alkyl group can besubstituted by (C₃-C₇)-cycloalkyl or can be mono- or polysubstituted byan aryl group, wherein the aryl group can be mono- or polysubstituted byhalogen, (C₁-C₆)-alkyl, (C₃-C₇)-cycloalkyl, hydroxy and nitro groups; R₁may be an aryl group, wherein the aryl group can be mono- orpolysubstituted by halogen, (C₁-C₆)-alkyl, (C₃-C₇)-cycloalkyl, hydroxyand nitro groups; R₁ may further be a protecting group;

R₂, R₃, R₄, R₅, R₆, and R₇ may be the same or different and are eachselected from the group consisting of hydrogen, (C₁-C₆)-alkyl,(C₃-C₇)-cycloalkyl, (C₁-C₆)-acyl, (C₁-C₆)-alkoxy, alkoxycarbonyl(COOR₁), halogen, benzyloxy, the nitro group, the amino group, the(C₁-C₄)-mono or dialkyl-substituted amino group, or an aryl group,wherein the aryl group can be mono- or polysubstituted by halogen,(C₁-C₆)-alkyl, (C₃-C₇)-cycloalkyl, hydroxy and nitro groups;

X and Z may be the same or different and are each selected from thegroup consisting of O, S and NH. The method comprises the step ofintroducing a double bond into the 5 membered ring of the4,5-dihydro-1,3-azoles moiety of a compound of the formula II

wherein the stereochemical centers may have R or S configuration.

In a preferred embodiment, preferably with MnO₂ or NiO₂.

In one embodiment, the compound of formula II is prepared by cyclizationof the derivatives of the N-substituted indole-3-glyoxylamide of formulaIII

where R₈ represents hydrogen or a protecting group.

In one embodiment, the compound of formula II is prepared by cyclizationof derivatives of the indole-3-glyoxylates of formula IV

where Z may be O or S, and R₈ represents hydrogen or the protectinggroup.

In one embodiment, the derivatives of the N-substitutedindole-3-glyoxylamide of formula III are obtained from derivatives ofindole-3-glyoxylic acid of formula V, and the corresponding amines offormula VI

where Y is selected from amino group, halogen, hydroxyl, alkoxy group(OR₁), mercapto group (SH) or alkylthio group (SR₁).

In another embodiment, derivatives of formula IV are obtained from thederivatives of indole-3-glyoxylic acid of formula V, and thecorresponding alcohols or thiols of formula VIII.

In another embodiment, the present invention is a method of synthesizingaromatic ketone compositions of formula I comprising the step ofintroducing two double bonds into the 5 membered ring of thetetrahydro-1,3-azoles of formula XI.

In a preferred embodiment, the compound of formula XI is prepared fromderivatives of the indole-3-glyoxals of formula X and the correspondingamines of formula VI.

In another embodiment, the present invention additionally comprising thestep of testing the compounds for efficacy as an AHR ligand.

In another embodiment, the present invention is a compound of theformula II

where R₁, R₂, R₃, R₄, R₅, R₆, R₇, X, and Z are as defined in claim 1,and where the stereochemical centers, i.e., the carbons bearing C(X)ZR₁and R₇ substituent may have R or S configuration.

In another embodment, the present invention is a compound of the formulaIII

wherein:

R₁, R₂, R₃, R₄, R₅, R₆, R₇, X, and Z are as defined in claim 1; R₈represents hydrogen or the protecting group; the stereochemical centers,i.e., the carbons bearing C(X)ZR₁ and R₇ substituent may have R or Sconfiguration; with the proviso that when R₇ and R₈ are hydrogens, and Xis an oxygen, Z can not be oxygen.

In another embodiment, the present invention is a compound of theformula IV

wherein:

R₁, R₂, R₃, R₄, R₅, R₆, X, and Z are as defined in claim 1; R₈represents hydrogen or the protecting group; Z may be O or S thestereochemical center, i.e., the carbon bearing NHR₈ substituent mayhave R or S configuration.

In another embodiment, the present invention is a compound of theformula XI

where R₁, R₂, R₃, R₄, R₅, R₆, R₇, X, and Z are as defined in claim 1,and where all the stereochemical centers of the tetrahydro-1,3-azolefragment may have R or S configuration.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING

FIG. 1 is a scheme describing a preferred embodiment of the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a process for preparing certainaromatic ketones possessing two heterocyclic moieties attached to acarbonyl group. One moiety comprises an indole (or substituted indole)fragment that is attached to the carbonyl group by carbon 3 of theindole. The other heterocyclic moiety comprises a five-membered1,3-azole ring of imidazole, oxazole or thiazole, attached to thecarbonyl group by the carbon 2 and possessing carboxyl group (free orderivatized) as substituent at carbon 4.

Compounds of the present invention are envisioned to have efficacy asAhR ligands. Characterization and function of the AhR ligand isdescribed in U.S. provisional patent application 60/268,809 and U.S.patent application Ser. No. 10/074,102, filed Feb. 12, 2002. Both ofthese applications are incorporated by reference as if set forth intheir entirety.

Thus, the present invention provides a process for preparing compoundsrepresented by the general formula I:

wherein:

R₁ may be hydrogen or can be selected from the group consisting of(C₁-C₆)-alkyl and (C₃-C₇)-cycloalkyl, wherein the alkyl group can besubstituted by (C₃-C₇) cycloalkyl, or can be mono- or polysubstituted bythe aryl group, wherein the aryl group can be mono- or polysubstitutedby halogen, (C₁-C₆)-alkyl, (C₃-C₇)-cycloalkyl, hydroxy and nitro groups,

R₁ may be an aryl group, wherein the aryl group can be mono- orpolysubstituted by halogen, (C₁-C₆)-alkyl, (C₃-C₇)-cycloalkyl, hydroxyand nitro groups,

R₁ may be a protecting group,

R₂, R₃, R₄, R₅, R₆, and R₇ may be the same or different and are selectedfrom the group consisting of hydrogen, (C₁-C₆)-alkyl,(C₃-C₇)-cycloalkyl, (C₁-C₆)-acyl, (C₁-C₆)-alkoxy, alkoxycarbonyl(COOR₁), halogen, benzyloxy, the nitro group, the amino group, the(C₁-C₄)-mono and dialkyl-substituted amino group or an aryl group,wherein the aryl group may be mono- or poly-substituted by halogen,(C₁-C₆)-alkyl, (C₃-C₇)-cycloalkyl, hydroxy and nitro groups, and

X and Z may be the same or different and are selected from the groupconsisting of O, S and NH.

In the method of the present invention, the compounds of formula I arepreferably prepared from indole derivatives of the general formula II:

where the stereochemical centers, i.e., the carbons bearing C(X)ZR₁ andR₇ substituent may have R or S configuration.

Such introduction of an additional double bond to the five membered ringof the 4,5-dihydro-1,3-azoles II can be a one-step dehydrogenation orcan comprise two steps. For example, allylicbromination/dehydrobromination procedures are suitable. In the firstcase, suitable reagents causing dehydrogenation of dihydro derivativesof nitrogen-, oxygen-, and sulfur-containing heterocycles are MnO₂(North and Pattenden, Tetrahedron 46:8267, 1990) and NiO₂ (Minster, etal., J. Org. Chem. 43:1624, 1978; Evans, et al., J. Org. Chem. 44:497,1979). However, other reagents can be satisfactorily employed for suchtransformation, including sulfur, oxygen, potassium ferricyanide,mercuric acetate, hydrogen peroxide, potassium dichromate, cupricsulfate, ferric chloride, phenanthrene-9,10-dione and other quinones.Examples of the methods which can be used for two-step conversion of IIto the fully aromatic compounds I involve a treatment with BrCCl₃ andDBU (Williams, et al., Tetrahedron Lett. 38:331, 1997; Freeman andPattenden, Tetrahedron Lett. 39:3251, 1998), NBS/hν (Meyers and Tavares,Tetrahedron Lett. 35:2481, 1994) and phenylselenenylation/elimination(Nakamura, et al., Tetrahedron Lett. 36:5059, 1995).

Compounds of the general formula II, in turn, can be prepared bycyclization of the derivatives of N-substituted indole-3-glyoxylamidesof the general formula III (where Z is NH, O or S) or derivatives ofindole-3-glyoxylates of the general formula IV (where Z is O or S):

where:

R₈ represents hydrogen or a protecting group.

It can be expected that acidic conditions, for example anhydroushydrochloric acid (McGowan, et al., J. Am. Chem. Soc. 99:8078, 1977) orstrong Lewis acid such as TiCl₄ (Walker and Heathcock, J. Org. Chem.57:5566, 1992) would be desirable for such cyclizations because theycould enhance a reactivity of C═X group for nucleophilic attack ofamino, hydroxy or thiol group. Presence of strong acids in the reactionmedium could also facilitate a subsequent dehydration process (whenX═O), evolution of H₂S (when X═S), or evolution of NH₃ (when X═NH) fromthe intermediates. The examples of other reagents which can be usefulfor such transformations are H₂SO₄, P₂O₅, PCl₅, SOCl₂ and Al₂O₃.Suitable solvents for such dehydrative cyclizations can be chlorinatedhydrocarbons such as chloroform or methylene chloride. It should beadded that, depending on the conditions of the reaction, suchcyclization can be successfully accomplished without prior removal ofS-, O- or N-protecting groups.

The derivatives of N-substituted indole-3-glyoxylamides of the generalformula III (where Z is NH, O or S) can be obtained from the derivativesof indole-3-glyoxylic acid V and the corresponding amines VI:

where:

Y is selected from amino group, halogen, hydroxyl, alkoxy group (OR₁),mercapto group (SH) or alkylthio group (SR₁).

For transformation of V to III conditions usually used for the synthesisof amides would be appropriate, such as, neutral solvent (for examplebenzene) and a presence of pyridine or tertiary amine (triethylamine,etc) as a catalyst. The compounds VI can be free amines or thecorresponding amine salts (hydrochlorides, etc). It should be emphasizedthat, depending on the nature of reagents and reaction conditions, thereaction between V and VI can directly provide cyclised compounds of thegeneral formula II. Such situation can take place, for example, withimino ethers V (where X═NH and Y═OR₁, see North and Pattenden,Tetrahedron 46:8267, 1990).

Derivatives of indole-3-glyoxylates of the general formula IV (where Zis O or S) can be obtained from the derivatives of indole-3-glyoxylicacid V and the corresponding alcohols (thiols) VII:

Conditions known by those of skill in the art as suitable for esterformation should be used.

The derivatives of indole-3-glyoxylic acid V are the known compounds orthey can be obtained by modification of the known methods. Thus, forexample, derivatives of 3-indoleglyoxylyl chlorides V (X═O, Y═Cl) can beefficiently obtained by acylation of the corresponding indoles withoxalyl chloride (Da Settimo, et al., Eur. J. Med. Chem. 23:21, 1988; J.Med. Chem. 39:5083, 1996). It should be emphasized that, depending onthe nature of reagents and reaction conditions, the reaction between Vand VII can directly provide cyclised compounds of the general formulaII.

Alternatively, the compounds of formula I can be prepared by cyclizationof the derivatives of N-substituted indole-3-glyoxylamides of thegeneral formula VIII or condensation of the derivatives ofindole-3-glyoxylamides of the general formula IX with the carbonylcompounds X:

where:

W can be halogen, hydroxyl or taken together with R₉ is a diazo (═N₂)group, and

R₉ is hydrogen or taken together with W is diazo (═N₂) group.

The cyclization process of VIII leading to compounds I is usually anacid-catalyzed reaction. Usually the condensation of IX and X requirestheir heating in an inert solvent, sometimes a presence of Lewis acidscan be also advantageous.

Alternatively, the preparation of the compounds of the general formula Ican be achieved by the ring synthesis of the tetrahydro-1,3-azoles ofthe general formula XI, i.e., the formation of N,N-, N,O- orN,S-analogues of cyclic acetals of the derivatives of theindole-3-glyoxals X and their subsequent dehydrogenation:

Such condensation process of X and VI usually requires heating of bothcompounds in an inert solvent. For dehydrogenation of thetetrahydro-1,3-azoles XI similar reagents and conditions can be appliedas in the case of conversion of 4,5-dihydro-1,3-azoles II to the fullyaromatic compounds I (see above).

In this specification and the claims, the term “protecting group” refersto any group commonly used for the protection of hydroxyl, thiol andamino functions during subsequent reactions. Such protecting groups arediscussed by T. W. Greene and P. G. M. Wuts in chapters 2, 6 and 7,respectively, of “Protective Groups in Organic Synthesis”, John Wileyand Sons, Inc., New York, 1999, incorporated herein by reference intheir entirety. The “hydroxyl-protecting groups” are acyl or alkylsilylgroups such as trimethylsilyl, triethylsilyl, t-butyldimethylsilyl andanalogous alkylated silyl radicals, or alkoxyalkyl groups such asmethoxymethyl, ethoxymethyl, methoxyethoxymethyl, tetrahydrofuranyl ortetrahydropyranyl. The “thiol-protecting groups” are alkyl or arylalkylgroups such as t-butyl, benzyl, adamantyl, cyanoethyl, etc. or acylgroups such as acetyl, benzoyl, trifluoroacetyl, etc. The“amino-protecting groups” are, for example, alkyl carbamates such asallyl carbamate (Alloc), t-butyl carbamate (BOC) and benzyl carbamate(Cbz) or amines can be protected as the corresponding amides such aschloroacetamide, trifluoroacetamide (TFA), etc.

“Alkyl” signifies a straight-chain or branched hydrocarbon radical of 1to 6 carbons in all its isomeric forms, such as methyl, ethyl, propyl,isopropyl, butyl, isobutyl, pentyl, etc., and the term “cycloalkyl” isunderstood as meaning radicals such as, for example; cyclopropyl,cyclobutyl, cycloheksyl, etc. The designation “alkoxy group” representsradicals such as, for example, methoxy, ethoxy, butoxy, etc. The term“acyl” means an alkanoyl group of 1 to 6 carbons in all its isomericforms, such as formyl, acetyl, propionyl, etc. or an aroyl group, suchas benzoyl, nitrobenzoyl or halobenzoyl, or a dicarboxylic acyl groupsuch as oxalyl, malonyl, succinoyl, glutaroyl, or adipoyl. The term“aryl” signifies a phenyl-, or an alkyl-, halo-, nitro- orhydroxy-substituted phenyl group. The designation “halogen” representsfluorine, chlorine, bromine or iodine.

This invention is described by the following illustrative examples. Inthese examples specific products identified by Arabic numerals (e.g. 1,2, 3, etc) refer to the specific structures so identified in the SchemeI in FIG. 1.

EXAMPLES

The endogenous ligand for the aryl hydrocarbon receptor (AHR),2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE,5, Scheme 1), has been isolated in very small quantities (ca. 20 μg) andidentified through extensive spectral studies. Given the biologicalimportance of the ligand, its chemical synthesis was obviously necessaryfor confirmation of the structural assignment and preparation of largeramounts of compound needed for studies of its physiological activity.Since the molecule of this aromatic ketone consists of two heterocyclicfragments, indole and 4-carbomethoxythiazole attached to the carbonylgroup, we sought possible synthetic routes involving intermediate indoleglyoxylamides.

Thus, we decided to prepare the desired compound (5) from glyoxylamide(3). The latter compound was easily obtained by acylation of theL-cysteine methyl ester (2) with indoleglyoxylyl chloride (1) carriedout in the benzene solution containing triethylamine (Da Settimo, etal., J. Med. Chem. 39:5083, 1996). Next, we performed cyclization of theglyoxylamide (3) by employing reaction conditions used by Mann, et al.(Martin, et al., J. Chem. Soc. Perkin Trans. 1:2455, 1999) in theirpreparation of analogs of curacin A, i.e. treatment with TiCl₄ indichloromethane. This methodology allowed us to isolate the desiredthiazoline ester (4) in 25% yield. Finally, three different methods ofoxidation of thiazoline (4) were examined. Thus, treatment of 4 withMnO₂ or NiO₂ in dichloromethane provided the indolecarbonyl-thiazole (5)in satisfactory yields (88 and 75%, respectively). A mild method ofdehydrogenation described by Williams et al. (McGowan, et al.,Tetrahedron Lett. 38:331, 1997), i.e. the use of BrCCl₃ and DBU indichloromethane, was less efficient (ca. 40%).

We have found that the HPLC retention time (Song, et al., supra, 2002)and spectroscopic properties of the synthesized compound 5 are identicalin all respects to those of the endogenous AHR ligand isolated in ourlaboratory from pig lung. Its successful synthesis, therefore,unequivocally confirms the structure and allows for further biologicaltesting aimed at establishing its physiological role in livingorganisms.

Example 1

Preparation of(2R)-2-[2′-(1″H-indol-3″-yl)-2′-oxo-acetyloamino]-3-mercaptopropionicacid methyl ester (3). To a stirred suspension of indoleglyoxylylchloride 1 (2.07 g, 10 mmol) and L-cysteine methyl ester hydrochloride 2(2.57 g, 15 mmol) in a dry benzene (150 mL) was added dropwisetriethylamine (4.2 mL, 3.03 g, 30 mmol) with stirring at 0° C. Coolingbath was removed and the mixture was stirred at room temperature for 20hours and then refluxed for 2.5 hours. The warm solution was filteredand a precipitate washed with benzene. Filtrate was washed with sat.NaHCO₃ and water, dried (Na₂SO₄) and evaporated. The crystalline residuewas purified by flash chromatography. Elution with chloroform/methanol(99:1) gave pure compound 3 (1.68 g, 55%) that was crystallized frombenzene: m.p. 145-146° C.; [α]²² _(D)+193° (c 0.8, CHCl₃); UV (EtOH)λ_(max) 255.5 nm (ε 11,000), 266.0 nm (ε 9,600), 273.5 nm (ε 8,700),330.5 nm (ε 9,700); IR (KBr) 3358, 3262, 2952, 2941, 2538, 1750, 1744,1662, 1605, 1500, 1493, 1434, 1352, 1314, 1235, 1227, 1157, 1135, 744cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ 9.02 (1H, d, J=3.3 Hz, 2″-H), 8.84 (1H,m, NH _(indole)), 8.44 (1H, m, 4″-H), 8.25 (1H, br d, J=ca. 8 Hz, CONH),7.45 (1H, m, 7″-H), 7.35 (2H, br m, 5″- and 6″-H), 4.92 (1H, ˜dt, J=8.2,4.7 Hz, CHCO₂Me), 3.83 (3H, s, CO₂Me), 3.06 and 3.12 (each 1H, each˜ddd, J=14, 9, 4.7 Hz, CH ² SH), 1.50 (1H, t, J=9.0 Hz, CH₂SH); ¹H NMR(500 MHz, CD₃OD) δ 8.77 (1H, s, 2″-H), 8.30 (1H, m, 4″-H), 7.46 (1H, m,7″-H), 7.25 (2H, m, 5″- and 6″-H), 4.77 (1H, part X of ABX system,J=6.6, 4.8 Hz, CHCO₂Me), 3.78 (3H, s, CO₂Me), 3.08 (1H, part A of ABXsystem, J=14.1, 4.8 Hz, one of CH ² SH), 3.01 (1H, part B of ABX system,J=14.1, 6.6 Hz, one of CH ² SH); ¹³C NMR (125 MHz, CD₃OD) δ 181.85 (s,COCONH), 171.69 (s, COOMe), 165.27 (s, COCONH), 139.76 (d, C-2″), 137.98(s, C-7a″), 127.86 (s, C-3a″), 124.92, 123.93 and 122.99 (each d; C-4″,-5″ or -6″), 113.96 (s, C-3″), 113.14 (d, C-7″), 55.85 (d, CHCO₂Me),53.16 (q, CO₂ Me), 26.48 (t, CH₂SH); MS (EI) m/z (relative intensity) noM⁺, 273 (M⁺−SH, 0.5), 256 (2), 233 (2), 185 (2), 183 (2), 153 (19), 144(indole-C(O)—, 22), 136 (8), 115 (8), 107 (25), 91 (100), 77 (63), 59(51); MS (ESI) m/z 329.0568 (M⁺+Na), C₁₄H₁₄N₂O₄SNa requires 329.0572;Anal. (calcd for C₁₄H₁₄N₂O₄S): C, 54.89; H, 4.61; N, 9.15; S, 10.47.Found: C, 54.87; H, 4.65; N, 9.17; S, 10.40. The compound gave a singlepeak on HPLC (20% 2-propanol in hexane, 10 mm×25 cm Zorbax-Sil column, 4mL/min) at R_(v) 27 mL.

Example 2

Preparation of(4R)-2-(1′H-indole-3′-carbonyl)-4,5-dihydro-thiazole-4-carboxylic acidmethyl ester (4). To a stirred solution of indoleglyoxamide 3 (2.53 g,8.3 mmol) in anhydrous methylene chloride (300 mL) was added TiCl₄ (1 Msol. in CH₂Cl₂, 8.4 mL, 8.4 mmol) dropwise at room temperature. Themixture was then refluxed for 5 hours, cooled to room temperature,stirred overnight (16 hours) and quenched by an addition of saturatedNaHCO₃. The organic layer was washed with water, dried (MgSO₄) andevaporated. The residue was purified by flash chromatography. Elutionwith chloroform/methanol (99:1) gave pure compound 4 (0.6 g, 25%) thatwas crystallized from methanol/benzene: m.p. 190-191° C.; [α]²² _(D)+64°(c 0.5, CHCl₃); UV (EtOH) λ_(max) 261.0 nm (ε 8,900), 268.5 nm (ε9,200), 275.5 nm (ε 8,800), 333.0 nm (ε 8,200); IR (KBr) 3420, 3222,2956, 1748, 1604, 1597, 1580, 1514, 1488, 1458, 1432, 1314, 1233, 1212,1187, 1133, 1071, 1055, 805, 776, 752 cm⁻¹; ¹H NMR (500 MHz, CD₃COCD₃) δ11.27 (1H, m, NH), 8.82 (1H, d, J=3.2 Hz, 2′-H), 8.34 (1H, m, 4′-H),7.56 (1H, m, 7′-H), 7.27 (2H, m, 5′- and 6′-H), 5.59 (1H, part X of ABXsystem, ˜t, J=ca. 9.5 Hz, 4-H), 3.80 (3H, s, CO₂Me), 3.67 (1H, part A ofABX system, J=11.3, 10.1 Hz, one of 5-H₂), 3.58 (1H, part B of ABXsystem, J=11.3, 8.9 Hz, one of 5-H₂); ¹³C NMR (125 MHz, CD₃COCD₃)

179.71 (s, C═O), 173.88 (s, C-2), 171.14 (s, COOMe), 138.73 (d, C-2′),137.39 (s, C-7a′), 127.22 (s, C-3a′), 124.31, 123.32 and 122.45 (each d,C-4′, -5′ or -6′), 114.06 (s, C-3′), 112.92 (d, C-7′), 80.49 (d, C-4),52.65 (q, CO₂ Me), 33.62 (t, C-5); MS (EI) m/z (relative intensity) 288(M⁺, 29), 256 (M⁺−MeOH, 4), 236 (7), 229 (M⁺−CO₂Me, 6), 202 (4), 144(100), 137 (15), 116 (M⁺−C₆H₆O₃SN, 15), 95 (16), 81 (41), 69 (87); MS(ESI) m/z 311.0454 (M⁺+Na), C₁₄H₁₂N₂O₃SNa requires 311.0466; Anal.(calcd for C₁₄H₁₂N₂O₃S): C, 58.32; H, 4.20; N, 9.72; S, 11.12. Found: C,58.34; H, 4.09; N, 9.77; S, 10.82. The compound gave a single peak onHPLC (20% 2-propanol in hexane, 10 mm×25 cm Zorbax-Sil column, 4 mL/min)at R_(V) 39 mL.

Example 3

Preparation of 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acidmethyl ester (5). (a) Freshly activated manganese (IV) oxide (115 mg,1.3 mmol) was added to a solution of indole-thiazoline ketone 4 (38 mg,0.13 mmol) in anhydrous methylene chloride (30 mL). The resultingsuspension was stirred for 3 hours at room temperature and thenconcentrated. The residue was purified by column chromatography. Elutionwith chloroform/methanol (99:1) gave pure compound 5 (33 mg, 88%) thatwas crystallized from methanol: m.p. 234-235° C.; UV (EtOH) λ_(max)271.0 nm (ε 10,500), 278.0 nm (ε 11,400), 356.5 nm (ε 11,700); IR (KBr)3452, 3241, 3125, 2957, 2927, 1737, 1593, 1577, 1507, 1482, 1466, 1432,1338, 1234, 1206, 1202, 1128, 1113, 1102, 1063, 816, 782, 776, 755, 642cm⁻¹; ¹H NMR MHz, CD₃COCD₃) δ 11.33 (1H, m, NH), 9.30 (1H, s, 2′-H),8.70 (1H, s, 5-H), 8.44 (1H, m, 4′-H), 7.61 (1H, m, 7′-H), 7.30 (2H, m,5′- and 6′-H), 3.94 (3H, s, CO₂Me); ¹H NMR (500 MHz, CD₃OD) δ 9.25 (1H,s, 2′-H), 8.66 (1H, s, 5-H), 8.36 (1H, m, 4′-H), 7.51 (1H, m, 7′-H),7.28 (2H, m, 5′- and 6′-H), 3.99 (3H, s, CO₂Me); ¹H NMR (500 MHz,DMSO-d₆) δ 9.08 (1H, s, 2′-H), 8.86 (1H, m, 5-H), 8.30 (1H, m, 4′-H),7.59 (1H, m, 7′-H), 7.28 (2H, m, 5′- and 6′-H), 3.91 (3H, s, CO₂Me); ¹³CNMR (125 MHz, DMSO-d₆) δ 176.45 (s, C═O), 169.86 (s, C-2), 161.01 (s,CO₂Me), 146.89 (s, C-4), 137.98 (d, C-2′), 136.34 (s, C-7a′), 133.90 (d,C-5), 126.35 (s, C-3a′), 123.63, 122.70 and 121.40 (d, C-4′, -5′ or-6′), 112.72 (d, C-7′), 112.04 (s, C-3′), 52.35 (q, CO₂ Me); MS (EI) m/z(relative intensity) 286 (M⁺, 70), 144 (M⁺−C₅H₄O₂SN, 100), 116(M⁺−C₆H₄O₃SN, 17), 89 (14), 69 (18); MS (ESI) m/z 309.0297 (M⁺+Na),C₁₄H₁₀N₂O₃SNa requires 309.0310. Anal. (calcd for C₁₄H₁₀N₂O₃S): C,58.73; H, 3.52; N, 9.78; S, 11.20. Found: C, 58.83; H, 3.46; N, 9.94; S,11.12.

(b) Nickel peroxide (assay ˜30% active NiO₂) (120 mg, 1.3 mmol) wasadded to a solution of indole-thiazoline ketone 4 (38 mg, 0.13 mmol) inanhydrous methylene chloride (30 mL). The resulting suspension wasstirred for 19 hours at room temperature and then concentrated. Theresidue was purified by column chromatography. Elution withchloroform/methanol (99:1) gave pure product 5 (28 mg, 75%) that wascrystallized from methanol. The spectroscopic properties of thesynthesized compound were identical in all respects with those describedin EXAMPLE 3a.

(c) To a stirred suspension of indole-thiazoline ketone 4 (255 mg, 0.88mmol) in methylene chloride (30 mL) was added dropwise DBU (148 mg, 146μL, 0.98 mmol) at 0° C. The solution turned red and became clear. Thenbromotrichloromethane (178 mg, 88 μL, 0.90 mmol) was added dropwise. Theyellow solution was stirred for 4.5 hours at 0° C. and for 20 hours incold room (ca. 6° C.). Saturated NH₄Cl was added and phases separated.Organic layer was washed with water, dried (MgSO₄), and evaporated. Theresidue was redissolved in chloroform, applied on a silica Sep-Pak (5 g)and eluted with 1% MeOH in chloroform. The corresponding fractionscontaining indole-thiazole-ketone 5 were combined, evaporated and theresidue (156 mg) was subjected to HPLC (10% 2-propanol in hexane, 10mm×25 cm Zorbax Silica column, 4 mL/min) separation. The product 5collected at R_(V) 42 mL (104 mg, 40%). The spectroscopic properties ofthe synthesized compound were identical in all respects with thosedescribed in EXAMPLE 3a.

1. A method of synthesizing aromatic ketone compositions of formula I

wherein: R₁ may be hydrogen or can be selected from the group consistingof (C₁-C₆)-alkyl and (C₃-C₇)-cycloalkyl, and wherein the alkyl group canbe substituted by (C₃-C₇)-cycloalkyl or can be mono- or polysubstitutedby an aryl group, wherein the aryl group can be mono- or polysubstitutedby halogen, (C₁-C₆)-alkyl, (C₃-C₇)-cycloalkyl, hydroxy and nitro groups;R₁ may be an aryl group, wherein the aryl group can be mono- orpolysubstituted by halogen, (C₁-C₆)-alkyl, (C₃-C₇)-cycloalkyl, hydroxyand nitro groups; R₁ may further be a protecting group; R₂, R₃, R₄, R₅,R₆, and R₇ may be the same or different and are each selected from thegroup consisting of hydrogen, (C₁-C₆)-alkyl, (C₃-C₇)-cycloalkyl,(C₁-C₆)-acyl, (C₁-C₆)-alkoxy, alkoxycarbonyl (COOR₁), halogen,benzyloxy, the nitro group, the amino group, the (C₁-C₄)-mono ordialkyl-substituted amino group, or an aryl group, wherein the arylgroup can be mono- or polysubstituted by halogen, (C₁-C₆)-alkyl,(C₃-C₇)-cycloalkyl, hydroxy and nitro groups; X and Z may be the same ordifferent and are each selected from the group consisting of O, S andNH; comprising the step of introducing a double bond into the 5 memberedring of the 4,5-dihydro-1,3-azoles moiety of a compound of the formulaII

wherein the stereochemical centers may have R or S configuration.
 2. Themethod of claim 1, wherein the step of introducing a double bondcomprises oxidation of a compound of the formula II.
 3. The method ofclaim 2, wherein the oxidation comprises treatment of the selectedcompound with MnO₂ or NiO₂.
 4. The method of claim 1, wherein thecompound of formula II is prepared by cyclization of the derivatives ofthe N-substituted indole-3-glyoxylamide of formula III

where R₈ represents hydrogen or a protecting group.
 5. The method ofclaim 4, wherein the cyclization comprises treatment with TiCl₄.
 6. Themethod of claim 1, wherein the compound of formula II is prepared bycyclization of derivatives of the indole-3-glyoxylates of formula IV

where Z may be O or S, and R₈ represents hydrogen or the protectinggroup.
 7. The method of claim 4, wherein the derivatives of theN-substituted indole-3-glyoxylamide of formula III are obtained fromderivatives of indole-3-glyoxylic acid of formula V, and thecorresponding amines of formula VI

where Y is selected from amino group, halogen, hydroxyl, alkoxy group(OR₁) mercapto group (SH) or alkylthio group (SR₁).
 8. The method ofclaim 6, wherein derivatives of formula IV are obtained from thederivatives of indole-3-glyoxylic acid of formula V, and thecorresponding alcohols or thiols of formula VII.


9. A method of synthesizing aromatic ketone compositions of formula Icomprising the step of introducing two double bonds into the 5 memberedring of the tetrahydro-1,3-azoles of formula XI.


10. The method of claim 9, wherein the compound of formula XI isprepared from derivatives of the indole-3-glyoxals of formula X and thecorresponding amines of formula VI.


11. The method of claim 1 additionally comprising the step of testingthe compound for efficacy as an AHR ligand.
 12. The method of claim 9additionally comprising the step of testing the compound for efficacy asan AHR ligand.
 13. A compound of the formula II

where R₁, R₂, R₃, R₄, R₅, R₆, R₇, X, and Z are as defined in claim 1,and where the stereochemical centers, i.e., the carbons bearing C(X)ZR₁and R₇ substituent may have R or S configuration.
 14. A compound of theformula III

wherein: R₁, R₂, R₃, R₄, R₅, R₆, R₇, X, and Z are as defined in claim 1;R₈ represents hydrogen or the protecting group; the stereochemicalcenters, i.e., the carbons bearing C(X)ZR₁ and R₇ substituent may have Ror S configuration; with the proviso that when R₇ and R₈ are hydrogens,and X is an oxygen, Z can not be oxygen.
 15. A compound of the formulaIV

wherein: R₁, R₂, R₃, R₄, R₅, R₆, X, and Z are as defined in claim 1; R₈represents hydrogen or the protecting group; Z may be O or S; thestereochemical center, i.e., the carbon bearing NHR₈ substituent mayhave R or S configuration.
 16. A compound of the formula XI

where R₁, R₂, R₃, R₄, R₅, R₆, R₇, X, and Z are as defined in claim 1,and where all the stereochemical centers of the tetrahydro-1,3-azolefragment may have R or S configuration.